摘要
目的:阿尔茨海默病(Alzheimer’s disease,AD)的患病率在全球范围内不断增加,然而其发病机制尚不清楚。研究表明AD的进展与神经细胞凋亡密切相关。本研究旨在探讨miR-15a和Bag5在β-淀粉样蛋白(β-amyloid protein,Aβ)诱导的AD神经细胞凋亡中的作用及其具体机制。方法:通过给SD大鼠脑部注射Aβ42构建AD大鼠模型,并使用Aβ42处理SH-SY5Y细胞构建AD细胞模型。采用莫里斯(Morris)水迷宫实验检测大鼠的学习记忆能力;苏木精-伊红(hematoxylin and eosin,HE)染色检测大鼠脑组织的病理变化;尼氏(Nissl)染色检测脑组织中细胞形态以及数量变化;生物信息学分析筛选与Bag5相互作用的上游miRNA;四甲基偶氮唑盐(methyl thiazolyl tetrazolium,MTT)检测细胞增殖能力;流式细胞术检测细胞的凋亡率;实时反转录聚合酶链反应(real-time reverse transcription PCR,real-time RT-PCR)检测miR-15a和Bag5的mRNA水平;蛋白质印迹法检测Bag5、Bax和Caspase-3的蛋白质表达水平。分别采用miR-15a敲减或过表达载体以及Bag5敲减载体处理大鼠和细胞,萤光素酶报告实验验证miR-15a与Bag5之间的结合关系。结果:Morris水迷宫实验、HE染色和Nissl染色结果表明AD大鼠模型成功建立,且Aβ可诱导AD大鼠中神经细胞凋亡和抑制miR-15a表达。与正常细胞相比,Aβ处理可显著增加细胞凋亡率和Bag5的表达,降低细胞活力和miR-15a表达,过表达miR-15a会进一步加强Aβ对细胞增殖和凋亡的效果,而敲减miR-15a的表达则起到相反的作用(均P<0.01)。萤光素酶报告实验证明miR-15a与Bag5存在负向靶向关系。且与单独敲减Bag5相比,敲减miR-15a和Bag5共转染会显著提高细胞活力以及Bag5的表达,同时明显降低细胞的凋亡率以及Bax、Caspase-3的表达,体内实验也得到一致的结果(均P<0.01)。结论:Aβ可抑制miR-15a的表达,进而诱导Bag5表达,以激活Bag5对于Aβ引起凋亡的保护作用。
Objective:The prevalence of Alzheimer’s disease(AD)is increasing globally,however its pathogenesis is still unclear.The evidence showed that the progression of AD was closely related to the apoptosis of nerve cells.This study amis to explore the role and specific mechanism of miR-15a and Bag5 in the apoptosis of nerve cells induced by beta-amyloid protein(Aβ)in AD.Methods:The AD rat model was constructed by injecting Aβ42 into SD rat brain and the AD cell model was constructed by treating SH-SY5Y cells with Aβ42.The learning and memory ability of rats was detected by Morris Water Maze.Hematoxylin and eosin(HE)staining was used to detect the pathological changes of brain tissues.Nissl staining was used to detect the changes of cell morphology and number in brain tissues.The upstream miRNA that interacted with Bag5 were screened by bioinformatics analysis.Methyl thiazolyl tetrazolium(MTT)assay was used to detect cell proliferation.Flow cytometry was used to detect the apoptosis rate of cells.Real-time reverse transcription PCR(realtime RT-PCR)was used to detect the mRNA levels of miR-15a and Bag5.Western blotting was used to detect the protein expression levels of Bag5,Bax and Caspase-3.MiR-15a knockdown or overexpression vectors or Bag5 knockdown vectors were transfected into AD rat model and AD cell models,respectively.Luciferase reporter assay was used to verify the binding relationship between miR-15a and Bag5.Results:Morris Water Maze,HE staining and Nissl staining showed that the rat model of AD was established successfully,and Aβcould induce neuronal apoptosis and inhibit the expression of miR-15a in AD rats.Compared with normal cells,Aβtreatment significantly increased apoptosis rate and Bag5 expression,and weakened cell proliferation and miR-15a(all P<0.01).Overexpression of miR-15a further enhanced the effect of Aβon cell proliferation and apoptosis,while knockdown of miR-15a expression had the opposite effect(all P<0.01).Luciferase reporter assay confirmed that there was a negative targeting relationship between miR-15a and Bag5.Compared with Bag5 knockdown alone,the co-transfection of miR-15a inhibitor and si-Bag5 significantly increased the cell proliferation ability and mRNA and protein levels of Bag5,and significantly reduced the cell apoptosis rate and the expression of Bax and Caspase-3,animal studies have also shown consistent results(all P<0.01).Conclusion:Aβcan inhibit the expression of miR-15a,thereby inducing the expression of Bag5 and activating the protective mechanism of Bag5 against Aβinduced apoptosis.
作者
潘琼
胡馨予
郭科
PAN Qiong;HU Xinyu;GUO Ke(Department of Obstetrics and Gynecology,Third Xiangya Hospital,Central South University,Changsha 410013;Department of Neurology,Third Xiangya Hospital,Central South University,Changsha 410013,China)
出处
《中南大学学报（医学版）》
CAS
CSCD
北大核心
2024年第7期1109-1119,共11页
Journal of Central South University ：Medical Science
基金
湖南省自然科学基金(S2021JJMSXM2227,2022JJ30898)。
